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Nucleic Acids Research, 1992, Vol. 20, No. 13 3419-3425
© 1992

MOLECULAR BIOLOGY

Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability

Heidi Major Sleister, Kathleen A. Mills1, Sue E. Blackwell, Ann M. Killary2, Jeffrey C. Murray1 and Robert E. Malone*

Department of Biological Sciences, University of Iowa Iowa City, IA 52242, USA 1Department of Pediatrics, University of Iowa Hospitals Iowa City, IA 52242, USA 2University of Texas M.D.Anderson Cancer Center, Division of Laboratory Medicine Houston, TX 77030, USA

* To whom correspondence should be addressed

Received March 17, 1992. Revised May 28, 1992. Accepted May 28, 1992.

In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.


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